Fertilizer type and humic acid improve the growth responses, nutrient uptake, and essential oil content on Coriandrum sativum L.

In recent decades, the over-use of chemical fertilizers has imposed many environmental challenges worldwide. Nowadays, organic fertilizers such as vermicompost and livestock manure have gained a huge interest in sustainable agricultural systems. A 2-year field research was conducted as factorial based on a randomized complete block design to assay the fertilizer and humic acid (HA) efficiency on the growth responses and essential oil composition of Coriandrum sativum. The treatments were different fertilizer sources (livestock manure, vermicompost, and chemical fertilizers) and humic acid fertigation before and at the beginning of the flowering stage. The highest protein content was observed under vermicompost × HA application before flowering (0.118 μmol L−1 and 0.128 μmol L−1, respectively). Moreover, the co-application of organic fertilizers × HA at the beginning of flowering resulted in a significant increase in the photosynthetic pigments and N, P, K, Fe, Zn, and Mn content. According to the GC-FID and GC–MS analysis, linalool (55.91–63.19%), γ-terpinene (4.65–6.13%), α-pinene (2.64–5.74%), geranyl acetate (3.49–5.51%), 2-dodecanal (2.92–4.46%), menthol (1.33–3.90%), p-cymene (1.73–2.24%), and geraniol (1.25–2.15%) were the main essential oil constituents. The top linalool content was obtained by using chemical fertilizers and vermicompost × HA at the flowering onset stage. In general, the results revealed that chemical fertilizers could be replaced with vermicompost × HA and their co-application positively influenced the growth responses and the essential oil composition of coriander. Furthermore, the results obtained would be advisable to the extension section and the pioneer farmers to amend the large-scale production systems in favor of environmental health.

Photosynthetic pigments content. The results revealed that the various fertilizers source × HA significantly improved chlorophyll a (Chl a) content ( Table 1). The highest content of Chl a was obtained in the vermicompost × HA before flowering (35.17 mg kg −1 FW), and in the application of chemical fertilizer × HA at the beginning of flowering (34.56 mg kg −1 FW), which were 43.31% and 40.83% higher than control, respectively (Fig. 3a). The lowest Chl a content was recorded in control (24.54 mg kg −1 FW), which had a non-significant difference with the application of HA before (23.3 mg kg −1 FW) and at the beginning (22.98 mg kg −1 FW) of flowering.
The findings showed that the different fertilizers source, HA, and their interaction significantly affected coriander plants' chlorophyll b (Chl b) content ( Table 1). The highest Chl b content (26.24 mg kg −1 FW) was obtained in livestock manure × HA before flowering, which showed an increase of up to 61.71% compared to the control (15.32 mg kg −1 FW). The lowest Chl b content was recorded in control and HA treatments before flowering (Fig. 3b). www.nature.com/scientificreports/ Moreover, the effects of various fertilizers sources, HA, and their co-application were significant on the carotenoids (CARs) content ( Table 1). The highest CARs content (20.8 g m −2 ) was observed in the application of vermicompost × HA at the beginning of flowering, which was 101.55% higher than control. The lowest content of CARs was traced in control (10.32 g m −2 ) and HA (9.95 g m −2 ) before the flowering stage (Fig. 4c).

Total soluble proteins (TSP) content. The results revealed that different fertilizers and their interaction
with HA significantly increased TSP, but HA did not affect the trait ( Table 1). The highest TSP was achieved with vermicompost × HA before flowering (0.128 mg g −1 FW), which was 149.51% more than control. The least TSP content was detected in control (0.0513 mg g −1 FW) and HA (0.0646 mg g −1 FW) before flowering and at the beginning (0.546 mg g −1 FW) of flowering (Fig. 4a).
Essential oil yield (EOY). The effects of different fertilizers type, HA, and their combinations were significant on the EOY (Table 1). Chemical fertilizer × without HA, chemical fertilizer × HA at the beginning of flowering and vermicompost × HA at the beginning of flowering led to the highest EOY (0.720 g m −2 , 0.692 g m −2 and 0.671 g m −2 , respectively). While, the least EOY was recorded in control with 0.288 g m −2 (Fig. 4c). The coapplication of various fertilizers and HA improved EOY up to 132-150% over the control.  www.nature.com/scientificreports/ Essential oil constituents. Twenty seven constituents were identified, accounting for 90-97% of the total essential oil. Linalool (63.99%) was the predominant constituents of coriander seed EO. In addition, γ-terpinene (6.13%), α-pinene (5.74%), geranyl acetate (5.51%), dodecanal (4.46%), menthol (3.90%), p-cymene (2.24%), and geraniol (2.15%) were identified as the other predominant constituents ( Table 2). The highest hydrocarbons and oxygenated monoterpenes were observed in the chemical fertilizer × HA application before and at the beginning of flowering, respectively (Table 3). At the beginning of flowering, the highest content of sesquiterpene hydrocarbons was recorded in the no-fertilizer × HA treatment (Table 4).

Macro-and micro-nutrients content.
Based on the results, the macro-and micro-nutrients contents were significantly affected by the different fertilizers source combined with HA. The highest content of N (3.73%), P (2.78%), and K (0.72%) were observed with the application of vermicompost × HA before flowering. The highest content of Fe (0.685 mg g −1 DM), Zn (0.189 mg g −1 DM), and Mg (0.119 mg g −1 DM) was obtained by using livestock manure × HA before flowering. The lowest content of macro-and micronutrients were noted in the control (Table 3).

Discussion
This experiment revealed that organic and chemical fertilizers combined with HA could improve the coriander plant height (Fig. 1a). Studies have shown that the application of organic fertilizer improved soil fertility and microbial flora structure 18 . Vermicompost is an organic fertilizer is a rich source of nutrients, such as N, P, and K, and through the release of some organic acids (e.g., oxalic acid) and by affecting the plant cells metabolism improves the nutrients uptake, photosynthetic activity and metabolic processes of several enzymes and hence influence the plants growth and height 19,20 . Similarly, a study on mint 20 is consistent with our results. In agreement www.nature.com/scientificreports/ with our findings, it has been reported that HA can directly improve plant growth by accelerating proteins synthesis, increasing water and nutrient uptake, and enhancing fertilizer use efficiency 21,22 .
In this experiment, lateral branch number was enhanced using different fertilizers source combined with HA ( Fig. 1b). Various studies suggested that applying organic fertilizers such as vermicompost and manure improves nutrients availability and root access to the minerals 23 . Thereby, these fertilizers indirectly increase the photosynthetic rate by developing the root system. As a result, more photo-assimilates are stored in the stem, leading to the production of more lateral branch number 24 . In line with our result, a study on coriander indicated that the highest recorded number of lateral stems was obtained by applying organic fertilizers 25 . According to the results, BY and plant DW of the coriander plants increased by using organic and chemical fertilizers × HA, although vermicompost combined with HA improved BY and plant DW more than other treatments (Figs. 1c, 2a). Vermicompost improves the plant quality and yiled by enhancing nutrient availability, mainly Fe and Zn adsorption 26 . Asadi et al. 12 reported that vermicompost in peppermint significantly increased BY in line with our findings. Additionally, growth stimulants, such as HA induced the biosynthesis of amino acids and ultimately improved BY with the regulation and activation of the proteins' metabolic pathways and enzymes activity 27 . The higher data for lateral branch number in this experiment could be due to the positive role of HA on the metabolism of the plant root system, physiological processes, photosynthetic rate, its hormonal effects, which finally improve BY and plant DW 28 . The same effect of organic fertilizer and HA application was also observed on Lens culinaris 29 , Orthosiphon stamineus Benth 30 and Lycopersicum esculentum 31 .
The use of chemical and organic fertilizers with high nitrogen content leads to photosynthetic organ development, increasing the production and storage of photo-assimilates 32 . Our results revealed that TSW positively correlated with the GY (Table 5). N availability is essential for flowering and pollination and even transferring assimilates and filling seeds. So, nitrogen-containing fertilizers play a crucial role in the number and yield of seeds. Moreover, the TSW was correlated to the nitrogen available during the growth stage 33 . Consistent with the current experiment, it has been reported that HA increased the TSW in canola 34 . Moreover, vermicompost can also increase N, P, and K content in the soil and promotes plant growth and yield 35 .
Chemical and organic fertilizers boost the content of the photosynthetic pigment by enhancing nitrogen absorption, increasing the light acquisition, assimilating production, and improving growth and yield 36 . Furthermore, the high photosynthesis potential under organic fertilizers is probably due to the stimulated activity of beneficial soil microorganisms, enhancing chlorophyll content in the plants 37 . It was found that photosynthesis pigments content was increased by using the organic and chemical fertilizers in combination with HA ( Fig. 3a-c). www.nature.com/scientificreports/ Similarly, it has been stated that the application of vermicompost significantly increased the contents of chlorophylls and carotenoids in Borago officinalis 38 and Lactuca sativa 39,40 . HA facilitates the transfer of nutrients through the chelation and reduces evapotranspiration 41 . Ali et al. 41 in sorghum and Mahmood et al. 42 in corn also observed that HA application increased the content of chlorophylls.
Several studies have pointed out the role of chemical and organic fertilizers in increasing the protein content of plants. It has been reported that vermicompost increases the concentrations of N and K in plants 43 . The adequate N content in the soil resulted in the soluble protein content, and also TSP content was improved with enhancing N per unit area of the leaf 44 . K also plays an essential role in plant metabolism and is one of the critical components in protein synthesis 45 . The results indicated that vermicompost and HA could enhance the TSP in coriander plants (Fig. 4a). HA enhances protein synthesis through a number of biochemical mechanisms,  Table 1. Variance analysis for the growth traits, minerals content and essential oil content and yield of coriander plants in response to the chemical and organic fertilizers as well as humic acid application. *, ** and ns, significant at the 5% and 1% probability levels and non-significant, respectively. www.nature.com/scientificreports/ such as the adsorption of active ions 46 . Therefore, there may be another reason for increasing TSP in our study. Similar to our results, an enhancement of TSP content has been reported with vermicompost and HA in Chinese cabbage 47 , peanuts 22,48 , and sorghum 49 . According to the results, the co-application of organic fertilizers and HA positively influenced coriander GY and EOY (Figs. 2c, 4d). Organic fertilizers provide moisture, prepare the soil substrate for better root and shoot growth and improve the yield due to the supply of nutrients, such as N 50 . Fertilizers such as nitrogen are essential for the production of structural proteins, and also are needed for the overall growth, development and yield of plants 51 . In addition, vermicompost increases the grain growth and yield by improving the availability of certain nutrients, in particular, Fe and Zn, and in turn by a direct effect on the plant metabolism. The application of HA increased nutrient uptake, photosynthesis pigments content, plant growth, and GY (Fig. 2c). HA probably affects the activity of Rubisco enzyme, which positively influences the photosynthetic potential, absorption of macro-and micronutrients, activity of some enzymes, cell membrane permeability, and eventually improves GY 20 .
Essential oils belong to the terpenoids, a significant class of plant secondary metabolites. The synthesis of terpenes precursors i.e., isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) need ATP and NADPH as photosynthesis product. Thus, photosynthesis potential directly affects the production of EO. Moreover, CO 2 and glucose are the initial precursors in forming essential oils 52 . The different fertilizer sources and HA applications increased the EOC and EO components of Ocimum basilicum var. purple 53 , Arachis hypogaea L. 54 , and Salvia officinalis 13 , which agree with the present research ( Fig. 4b and Table 2). The use of organic fertilizers such as vermicompost and HA can also increase the EOC by enhancing the uptake of P and N, which are the major prerequisites for the primary and secondary metabolism in most medicinal plants 13,55 .
In addition, the highest concentration of micro-elements (Fe, Zn, and Mn) was obtained by applying vermicompost × HA before flowering ( Table 3). The different fertilizer sources improved micro-elements content due to enhancing the cation exchange capacity of the soil, the gradual release of nutrients, and the biological activities and physicochemical properties of the soil 56 . According to these results, an increase in the macro-elements (e.g., N, P, and K) has also been reported in peppermint plants treated with organic and chemical fertilizers 57 . The results also showed that HA foliar application significantly affected the content of macro-and microelements compared to the control.  T1  T2  T3  T4  T5  T6  T7  T8  T9  T10  T11  www.nature.com/scientificreports/  Table 4. Monoterpenes and sesquiterpenes percentage of coriander essential oil under the influence of organic and chemical fertilizers and humic acid (average of 2 years). T1 without fertilizer × without HA (control), T2 without fertilizer × HA before flowering, T3 without fertilizer × HA at the beginning of flowering, T4 vermicomposting × 0 HA, T5 vermicomposting × HA before flowering, T6 vermicomposting × HA at the beginning of flowering, T7 manure × without HA, T8 manure × HA before flowering, T9 manure × HA at the beginning of flowering, T10 chemical fertilizer × without HA, T11 chemical fertilizer × HA before flowering, T12 chemical fertilizer × HA at the beginning of flowering. Treatments   T1  T2  T3  T4  T5  T6  T7  T8  T9  T10  T11 Table 5. Pearson's correlation factors (coefficients) between total grain yield, essential oil content, essential oil yield, and chemical composition of coriander essential oil influenced by the chemical and organic fertilizers × humic acid application. *, **, Significant at 5 and 1% probability levels, respectively. -tailed). www.nature.com/scientificreports/ In conclusion, our study focused on the effect of the fertilizer source × HA on the nutrients absorption, growth responses, and the content, yield, and composition of coriander essential oil. It van be concluded from the present study that the application of vermicompost × HA fertigation before flowering improved the morphological trait, total soluble protein content, photosynthesis pigments and macro-and micro-nutrient contents. Furthermore, the chemical fertilizer and vermicompost × HA fertigation at the beginning of flowering increased the content and yield of coriander essential oil. The main idea of sustainable agricultural systems is to reduce the use of chemical fertilizers. The current experiment exhibited that chemical fertilizers could be replaced by vermicompost, livestock manure, and HA. The overall results could be advisable to the extension section, and the pioneer farmers to reduce the chemical fertilizers input and secure environmental health. However, many new studies are required for the evaluation of the efficiency of humic acid application and other organic fertilizer sources at different growth stages can be realized on a large scale.

Materials and methods
Study site and treatment. The Table 6. The soil was composed of sandy clay loam with pH 8.16, 1.23% organic carbon, 0.09% total N, 11.05 and 570.85 mg/kg of available P and K, respectively (depth of 0-30 cm). The climatic data in the research area are presented in Table 7.
This research was conducted as a factorial experiment based on a randomized complete block design (RCBD) with three replications. Experimental factors included fertilizer type and HA including: (T1) without fertilizers × without HA (control), (T2) without fertilizers × HA before flowering (200 mg L −1 ), (T3) without fertilizers × HA at the beginning of flowering (200 mg L −1 ), (T4) vermicompost (1.5 kg m −2 ) × without HA, (T5) vermicompost × HA before flowering (T6) vermicompost × HA at the beginning of flowering, (T7) manure (4 kg m −2 of livestock manure) × without HA, (T8) manure × HA before flowering (T9), manure × HA at the beginning of flowering (T10) chemical fertilizers (20 g m −2 of urea and 10 g m −2 of triple superphosphate) × without HA, (T11) chemical fertilizers × HA before flowering and (T12) chemical fertilizers × HA at the beginning of flowering. The humic acid fertigation was applied before flowering 60 days after sowing and was used 70 days after sowing for the beginning of flowering. The chemical fertilizers were applied according to the chemical and physical soil analysis. Humic acid was prepared from Humic Miracle, whose chemical characteristics are presented in Table 8. The livestock manure, vermicompost, and chemical fertilizers were applied during farm preparation.
The landrace coriander seeds were collected from East Azerbaijan Province. In May of 2018 and 2019, 36 experimental plots were prepared. Vermicompost and livestock manure were added one month before planting and triple superphosphate at the planting time, but urea was applied 20 and 40 days after sowing as top-dressing. Each plot (2 × 3 m 2 ) consisted of 7 rows with 35 cm row distance and 15 cm within rows. After planting the seeds, the plots were irrigated by the drip irrigation method. Operations such as irrigation, weed control, etc. were performed regularly during the growing season. All agronomic practices were performed uniformly for all plots.  www.nature.com/scientificreports/ Measurement of growth parameters. After full maturity (110-120 days after sowing), all experimental plants were separately harvested, and then yield, and its components were recorded. Traits such as plant height, stem diameter, shoot dry matter (DM), biological yield (BY), thousand-seeds weight (TSW), and grain yield (GY) were recorded on five plants per plot at the physiological maturity and harvest time.
Photosynthetic pigments content. Chlorophylls (Chl a and b) and carotenoids (CARs) content were determined spectrophotometrically using equations described by the Arnon method 58 . Leaf sample (0.5 g) was ground by liquid nitrogen and was suspended in 10 ml of %80 acetone. Their content was determined by measuring the extinction of the extract at the significant red absorption maxima of Chl a (664 nm) and b (647 nm) and CARs (470) and inserting these values simultaneously into the following Eqs. (1, 2 and 3 Essential oil distillation and analysis. The harvested seeds (40 g) were hydro-distilled to determine the percentage of essential oil using the Clevenger of British Pharmacopoeia for 3 h. Anhydrous sodium sulfate was added to each distillated essential oil and then stored at 4 °C before analysis to remove the water droplets. The essential oil content and yield were calculated using the following formulas: GC-MS analysis. The essential oils were analyzed using GC-FID and GC-MS. The analysis was conducted using an Agilent 7990 B gas chromatograph equipped with a 5988A mass spectrometer and a HP-5MS (0.25 mm i.d., 30 mL, 0.25 μm f.t., 5% phenyl methyl polysiloxane). The following oven temperature was used: 5 min at 60 °C, then up to 240 °C with the rate of 3 °C min −1 , held for 10 min. Helium (carrier gas) flow rate was 1 mL min −1 ; the injector split ratio was 1:30; the mass range and electron impact (EI) were 400 m/z and 70 eV, respectively. The identification of constituents was performed using the procedure explained by Morshedloo et al. 60 , which is based on the interactive combination of linear retention indices (RIs), calculated respect to a homologous series of n-alkanes (Supelco, Bellefonte, CA), and the mass spectrum (MS) matching with commercial libraries (ADAMS, WILEY 275 and NIST 17). GC-FID analysis was performed using an Agilent 7990 B gas chromatograph equipped with a flame ionization detector (FID), capillary column VF 5MS (30 mL, 0.25 mm i.d., 0.50 μm f.t., 5% phenyl methyl polysiloxane). The same oven temperature reported for GC-MS was used. The injection volume of the essential oil was 1 μL the essential oil in n-hexane (1:100). Quantification of the constituents was performed by peak area normalization without using correction factors 61 .
Statistical analysis. All data were subjected to a normality test via the Anderson-Darling method, and homogeneity of data was checked through Levene's test. Then, data were subjected to combined ANOVA using MSTAT-C Software. The significant differences among means were compared with the Last Significant Difference (LSD) test at P < 0.05. Pearson's correlation coefficient was calculated between grain yield, essential oil content, essential oil yield, and significant constituents of coriander essential oil. Since the effect of time (year) in the combined analysis of the experiment was not significant, the average data of two years were analyzed as a factorial experiment based on a randomized complete block design (RCBD). Essential oil content (%) = distillated essential oil g /40 g × 100, Essential oil yield g m −2 = mass of distillated essential oil g mass of grain yield g × 100.